The ability of cells to keep time over approximately 24 hours owes to an endogenous molecular oscillator known as the circadian clock, whose rhythms persist in the absence of environmental cues. The circadian clock regulates a range of metabolic, physiological, and behavioral activities in all higher eukaryotes. To protect the core oscillator from spurious resetting signals, the clock uses a mechanism called "gating" to buffer input cues in a phase-dependent manner. Loss of EARLY FLOWERING 3 (ELF3) function in Arabidopsis thaliana renders the core oscillator hypersensitive to resetting light signals. This investigation aims understand at the molecular level how ELF3 gates light entrainment of the Arabidopsis circadian clock. Our hypothesis is that ELF3 exerts its effects by interacting with other proteins and altering their activity. The specific aims of this proposal are to: 1) Determine whether physical interaction with phytochrome B (phyB) is critical to ELF3 function. Previous work has demonstrated that ELF3 directly interacts with the red light photoreceptor phyB in vitro, however, this interaction remains largely uncharacterized. Mutants in ELF3 lacking the capacity to interact with phyB, called interaction defective ELF3 (ELFSdi), will be constructed and introduced into elf3 plants to assay for complementation of the mutant phenotype. 2) Identify novel ELF3 interacting partners. Potential molecular partners of ELF3 will be identified using yeast two-hybrid by screening. Novel interacting clones will be confirmed by co-immunoprecipitation assays, their relationships to ELF3 and the core oscillator will be investigated in plants by molecular and genetic analyses. 3) Identify genes required for the elf3 circadian phenotype using a suppressor screen. EMS mutagenized elf3-1 plants will be screened for partial or complete restoration of clock function. The phenotypes of putative suppressors will be characterized using a battery of assays aimed at circadian clockrelated phenotypes, and the mutant locus will be identified with recently developed mapping protocols making use of high density oligonucleotide arrays. Relevance: The circadian clock regulates numerous important processes in eukaryotic organisms. That a defective clock can predispose humans to depression, obesity, abnormal sleep patterns, and certain types of cancer emphasizes the importance of proper time keeping. This research will investigate how circadian processes are regulated at the molecular level in eukaryotic cells. [unreadable] [unreadable] [unreadable]